Secondary anti mouse hrp antibody

Manufactured by Santa Cruz Biotechnology

Sourced in Belgium

The secondary anti-mouse HRP antibody is a laboratory reagent used to detect and quantify mouse proteins in various immunoassays. It binds to the primary mouse antibody and is conjugated with the enzyme horseradish peroxidase (HRP), which can be used to generate a colorimetric or chemiluminescent signal for visualization and measurement purposes.

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Lab products found in correlation

Spectramax m2 plate reader, by Molecular Devices (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Mini gel apparatus, by Bio-Rad (1 mentions) Lumi light detection kit, by Roche (1 mentions) Mouse monoclonal anti alpha tubulin antibody, by Merck Group (1 mentions) Imagequant las 4000 system, by GE Healthcare (1 mentions) Monoclonal mouse antibody, by Santa Cruz Biotechnology (1 mentions) Monoclonal mouse antibody, by Thermo Fisher Scientific (1 mentions) Ecl prime kit, by Cytiva (1 mentions) M per lysis buffer, by Thermo Fisher Scientific (1 mentions)

4 protocols using secondary anti mouse hrp antibody

Detecting SMO-1 and MRG-1::3XHA in C. elegans

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Control and smo-1 RNAi-treated worms were collected and washed with M9 buffer to remove bacteria. Samples were frozen at -20°C in SDS/PAGE sample buffer. Right before loading, samples were boiled for 10 min to denature the proteins and centrifuged for 10 min at full speed. MRG-1::3XHA was detected with rat anti-HA-HRP antibody (Roche) at a dilution of 1:500, SMO-1 was detected with anti-SUMO 6F2s mouse monoclonal from Hybridoma bank, and the secondary anti-mouse HRP antibody (catalog no. sc-2005; Santa Cruz Biotechnology) at 1:5.000 dilution.
As a standard loading control, we used anti-alpha-tubulin mouse monoclonal antibody from Sigma at 1:10000 dilution, and the secondary anti-mouse HRP antibody (catalog no. sc-2005; Santa Cruz Biotechnology) at 1:5.000 dilution. The Lumi Light detection kit (Roche) and the ImageQuant LAS4000 system (GE Healthcare Life Sciences) were used for the signal detection.

Baytek G., Blume A., Demirel F.G., Bulut S., Mertins P., & Tursun B. (2021). SUMOylation of the chromodomain factor MRG-1 inC. elegansaffects chromatin-regulatory dynamics.

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Solid-State NCAM Binding Assay

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The affinity of NCAM to different substrates was evaluated using solid state binding assay. Different types of silk films and poly-lysine were coated onto Immulon 2 HB 96-well assay plates (Thermo Scientific) with method mentioned above but scaled down according to the surface areas. Plates were blocked by 1% BSA in PBS for 1 hr at RT. Recombinant NCAM (R&D Cat#2408-NC-050) was subsequently added to each well at a concentration of 10 μg/ml and incubated for 1 hrs. Bound NCAM on silk films were detected by mouse anti-human NCAM antibody (1:500, R&D) for 1 hr. Secondary anti-mouse HRP antibody (1:3000, Santa Cruz) was incubated for 1 hr and 50 μl of TMB (3,30,5,50-tetramethylbenzidine) solution (Invitrogen) was added for the colorimetric reaction. Plates were washed by 150 μl of PBST for 6 times between every step. Color was allowed to develop at room temperature for 10 min and 50 μl of 1 N HCl was added to stop the reaction. OD_450nm was recorded from the 96-well plate using a Spectra Max M2 plate reader (Molecular Devices, Sunnyvale, CA) for data analysis. Appropriate controls included NCAM directly coated onto plates (+) and uncoated plates blocked by BSA (−). All assays were performed in triplicates.

An B., Tang-Schomer M., Huang W., He J., Jones J., Lewis R.V, & Kaplan D.L. (2015). Physical and Biological Regulation of Neuron Regenerative Growth and Network Formation on Recombinant Dragline Silks. Biomaterials, 48, 137-146.

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Western Blot Protein Detection

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Proteins were separated on 10% acrylamide gels in a Biorad mini gel apparatus. For Western blots, the proteins from the gel were transferred onto PVDF-membrane (Millipore) by tank blotting electro transfer. Western blots were performed by first blocking free binding sites on the membrane with 5% milk powder in TBST for 1 h. The blots were washed with TBST and incubated with the monoclonal antibodies, diluted 1:50 in TBST-5% BSA overnight in a cold room. Blots were washed 3 times for 10 min each with TBST and thereafter incubated with a secondary anti-mouse-HRP antibody (Santa Cruz SC-2005; 1:7500 dilutions in TBST with 0.5% BSA) for 1 h at room temperature. After washing the blot three times 10 min each in TBST, the chemiluminescence reaction was started. Images were recorded on a Fuji-LAS3000 cooled camera system.

Kohlberger M., Thalhamer T., Weiss R, & Tenhaken R. (2018). Arabidopsis MAP-Kinase 3 Phosphorylates UDP-Glucose Dehydrogenase: a Key Enzyme Providing UDP-Sugar for Cell Wall Biosynthesis. Plant Molecular Biology Reporter, 36(5), 870-877.

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Measuring HMG-CoA Reductase in Fluvastatin-Resistant Cells

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WT and fluvastatin-resistant replicon containing cells were cultured in the absence or presence of 5 µM fluvastatin for 24h. Cells were lysed in M-PER lysis buffer (ThermoScientific, Erembodegem, Belgium), denatured and loaded onto a 4-20% SDS-PAGE gel. After separation and transfer to a PVDF membrane, immunoblotting was performed to detect HMG-CoA reductase using a monoclonal mouse antibody
(1:100, Santa Cruz, Heidelberg, Germany) and β-actin as endogenous control with a monoclonal mouse antibody (1:10000, ThermoScientific, Erembodegem, Belgium).
The secondary anti-mouse HRP antibody (Santa Cruz, Heidelberg, Germany) was used at a dilution of 1:1000. Bound antibodies were visualized using the ECL Prime kit (Amersham, Diegem, Belgium) and the BioRad imaging system.

Delang L., Scheers E., Grabner M., Verpaalen B., Helsen N., Vanstreels E., Daelemans D., Verfaillie C, & Neyts J. (2015). Understanding the molecular mechanism of host-based statin resistance in hepatitis C virus replicon containing cells. Biochemical pharmacology, 96(3).

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